TRANSMISSION OF INTESTINAL BLASTOCYSTOSIS RELATED TO THE QUALITY OF DRINKING WATER

SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH TRANSMISSION OF INTESTINAL BLASTOCYSTOSIS RELATED TO THE QUALITY OF DRINKING WATER Paanjit Taamasri 1, Mathirut Mungthin 1 Ram Rangsin 2, Benchamart Tongupprakarn 1, Wirote Areekul 2 and Saovanee Leelayoova 1 1 Department of Parasitology and 2 Department of Military and Community Medicine, Phramongkutklao College of Medicine, 315 Rajavithi Road, Rajathevi, Bangkok 10400, Thailand Abstract. A cross-sectional study was performed to evaluate the risk factors of Blastocystis hominis infection in the Thai army population of the 11 th Infantry Division, Chachoengsao Province, Thailand. 201 army personnel and their family members were enrolled in this study. Intestinal parasitic infections in this population were assessed by stool examination using simple smear, formalin/ether technique and Kato-thick smear. Approximately one third of the specimens were positive for one or more intestinal parasites. With the prevalence of 21.9%, B. hominis was the most common intestinal parasite found in this population. Our data indicated that blastocystosis in this army population was significantly linked to the quality of drinking water. After being adjusted for potential confounders, consuming neither filtered nor boiled water was independently associated with blastocystosis. INTRODUCTION Blastocystis hominis is one of the most common intestinal protozoa in humans (Ashford and Atkinson, 1992; Zierdt et al, 1995). Having a worldwide distribution, B. hominis infection appears in both immunocompetent and immunocompromised individuals (Henry et al, 1986; Laughon et al, 1988; Church et al, 1992; Cegielski et al, 1993). Although several reports have suggested that B. hominis could cause gastrointestinal disorders (Sheehan et al, 1986; Kain et al 1987; Hussain Qadri et al, 1989; Doyle et al, 1990; Zierdt, 1991; Nimri 1993; Nimri and Batchoun, 1994), the certain pathogenicity of this organism need to be defined. The clinical consequence of B. hominis infection is mainly diarrhea or abdominal pain with other nonspecific gastrointestinal symptoms such as nausea, anorexia, vomiting, weight loss, lassitude, dizziness and flatulence (Sheehan et al, 1986; Kain et al 1987; Hussain Qadri et al, 1989; Doyle et al, 1990; Sinniah and Rajeswari, 1994; Shlim et al, 1995). Although the life cycle of B. hominis has been proposed (Singh, 1995), its validation has to be confirmed experimentally. B. hominis infection is usually diagnosed by light microscopic examination of stained smears or wet mounts of fecal material. In general, three morphological forms of B. hominis (vacuolar, granular, and ameboid) have Correspondence: Tel/Fax: 66-2-245-8331 been recognized. Although additional forms: cyst, avacuolar and multivacuolar (Zaman, 1994; MacPherson and MacQueen, 1994; Zaman, 1996) have been recently identified, most laboratories identify B. hominis by observing the vacuolar form. The transmissionable form of this organism has not been defined yet. It has been speculated that thick-walled cysts might be responsible for external transmission while thin-walled cysts might reinfect within a host s intestinal tract (Singh 1995). As with other intestinal protozoa, it is assumed that B. hominis is transmitted by the fecal-oral route (Kain et al, 1987; Zierdt, 1991; Boreham and Stenzel 1993; Nimri and Batchoun 1994; Singh et al, 1995). Although there have been several studies which suggested that B. hominis could be transmitted via untreated water (Kain, 1987; Nimri, 1993; Nimri and Batchoun, 1994), a significant association has not been confirmed yet. To evaluate the association between blastocystosis and quality of water, a cross-sectional study was conducted at the army base in Chachoengsao, a province of Thailand where B. hominis was commonly detected. MATERIALS AND METHODS The study was conducted in 1998 at the 11 th Infantry Division, Chachoengsao, a province situated nearby Bangkok, Thailand. The whole population in this army division was 1293; of these 689, 112 Vol 31 No. 1 March 2000

BLASTOCYSTOSIS IN THAILAND 437, 70 and 97 were privates, non-commissioned officers, commissioned officers and family members, respectively. The army members routinely exercise within the area of this army division. While most of the officers and their families were local people, almost all privates in this division came from northeastern Thailand and had to serve in the army for 2 years. The majority of this community were privates in the age range 20 to 30 years. There were 201 members of this army community who voluntarily enrolled in this study. The water supply of the 11 th Infantry Division was mainly tap water and rain water with or without further treatment. Privates were living together in the buildings and consuming water that was available for them, either tap or rain water, while officers and their families were living in separate houses and able to choose any source of drinking water and also its treatment. Stool specimens were collected and examined immediately for B. hominis by simple smear preparation. Saline and lugol iodine preparation of each sample were examined under 10x and 40x magnification. Each stool sample was preserved in 10% formalin solution in order to perform a formalin/ ether concentration technique. Trichrome staining was also done to confirm the suspected specimens. Kato-thick smear technique was used to identify intestinal helminths in every specimen. To determine the risk factors of B. hominis infection in this community, individual was asked to fill the health assessment questionnaires concerning age, rank, sex, type of water consumed, history of diarrhea and other gastrointestinal symptoms. Individual data was compared to his/her result of stool examination. Univariate analysis was performed using Epi Info version 6.04b. Odds ratios with 95% confidence intervals and p-values were calculated to compare outcomes among study groups. Logistic regression using SPSS for Windows version 7.5.2 was performed for multivariate analysis to assess the independent association of risk factors and B. hominis infection. RESULTS Of 201 subjects who were enrolled in this study, 82 (40.8%), 68 (33.8%), 29 (14.4%) and 22 (10.9%) were privates, non-commissioned officers, commissioned officers and family members, respectively. Age range of the study population was between 2 to 68 years. Half of them were healthy young men 21 to 30 years of age and the majority of this group were privates. Less than 10% of the enrolled subjects were female since only the officers could stay in this army base with their families. 75 of 201 (37.3%) samples were found to be positive for one or more intestinal parasites and 13 (6.5%) of these samples were mixed infection. Prevalence of intestinal parasitic infections was 64.6% in private group and 18.5% in the others including officers and family members (Table1). By both simple smear and concentration technique, 4 species of intestinal protozoan were identified (Blastocystis hominis, Endolimax nana, Giardia intestinalis and Chilomastix mesnili) (Table 2); of these the most common intestinal protozoa in this study was B. hominis (21.9%). Intestinal helminths were also found in this population with 17.9% and 0.5% positive for hookworm and Opisthorchis viverrini, respectively. Table 1 Prevalence of intestinal parasitic infections in the members of the 11 th Infantry Division. Rank No. No. examined positive (%) Privates 82 53 (64.6) Non-commissioned officers 68 9 (13.2) Commissioned officers 29 6 (20.7) Family members and others 22 7 (31.8) Total 201 75 (37.3) Table 2 Intestinal parasites identified by simple smear and concentration techniques. Intestinal parasites No. positive (%) Protozoa: 52 (25.9) Blastocystis hominis 44 (21.9) Endolimax nana 5 (2.5) Giardia intestinalis 2 (1) Chilomastix mesnili 1 (0.5) Helminths: 37 (18.4) Hookworm 36 (17.9) Opisthorchis viverrini 1 (0.5) Vol 31 No. 1 March 2000 113

SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH Table 3 Characteristics of the enrolled subjects and prevalence of Blastocystis hominis infection. Characteristics Total No. No. of enrolled subjects B. hominis positive (%) Age group (yrs) <20 90 12 1 (8.33) 21-30 398 102 35 (34.3)* 31-40 501 50 6 (12) 41-50 213 22 1 (4.5) 51-60 78 13 1 (7.7) >60 7 2 0 Gender Male 1,259 184 43 (23.5) Female 28 17 1 (5.9) Rank Privates 689 82 32 (39)* Non-commissioned 437 68 5 (7.4) Commissioned 70 29 3 (10.3) Family member 97 22 4 (18.2) Total 1,287 201 44 (21.9) * Significant difference comparing to other groups in the same category (p < 0.05). Table 4 Univariate and multivariate analysis of the relationship between risk factors and B. hominis infection. Risk factor B. hominis B. hominis Crude OR p-value Adjusted OR + p-value positive (%) negative (%) (95%CI) (95%CI) Age (years) > 30 8 (18.2) 79 (50.3) 1 1 21-30 35 (79.5) 66 (42) 5.24 <0.0005 1.48 0.575 (2.12-13.21) (0.38-5.76) < 20 1 (2.3) 12 (7.6) 0.82 1.0 0.73 0.8 (0.02-7.17) (0.07-7.84) Gender Female 1 (2.3) 16 (10.2) 1 1 Male 43 (97.7) 141(89.8) 4.88 0.13 1.71 0.632 (0.71-209.3) (0.18-15.72) Rank Non-private 12 (27.3) 107 (68.2) 1 1 Private 32 (72.7) 50 (31.8) 5.71 <0.0005 3.04 0.072 (2.57-12.88) (0.91-10.23) Type of drinking water Boiled or filtered 6 (13.6) 88 (56) 1 1 Untreated 38 (86.4) 69 (43.9) 4.97 <0.0005 2.72 0.047 (1.92-15.1) (1.01-7.35) OR: Odds ratios, CI: Confidence interval, Odds ratios were adjusted for age (in 3 age groups), gender, rank and type of drinking water. 114 Vol 31 No. 1 March 2000

BLASTOCYSTOSIS IN THAILAND Table 3 shows the characteristics of the enrolled subjects and prevalence of blastocystosis in each group. The prevalence of blastocystosis was highest in the privates and in the age group 21 to 30 years old. By univariate analysis (Table 4), there were statistically significant associations between blastocystosis and the following risk factors: age between 21-30 years [OR, 5.24 (2.12-13.21)], being a private rank [OR, 5.71 (2.57-12.88)] and consuming neither boiled or filtered water, [OR, 4.97 (1.92-15.10)]. The multivariate logistic regression analysis showed that after adjusting for age, gender and rank, consuming neither boiled nor filtered water was independently associated with blastocystosis, adjusted OR, 2.72 (1.01-7.35). Thus after adjusting for potential confounders (age, gender and rank), those who consumed untreated water were 2.72 times more likely to develop blastocystosis than those who did not. DISCUSSION Although B. hominis infection has been reported from both developed and developing countries, higher prevalence is reported from developing countries, especially in tropical areas (Henry et al, 1986; Kain et al, 1987; Ashford and Atkinson, 1992; Church et al, 1992; Nimri, 1993). Prevalence of B. hominis infection in developing countries is between 30-50% which makes this organism one of the most common intestinal parasitic infections in human (Ashford and Atkinson 1992; Zierdt et al, 1995). B. hominis is also frequently found in westerners who traveled to tropical countries (Sheehan et al, 1986; Kain et al, 1987; Doyle et al, 1990; Shlim et al, 1995). In the present study, blastocystosis was the most common intestinal parasitic infection with the prevalence of 21.9%. Blastocystosis was found in the privates greater than that in the others. Hookworm was also highly detected in the privates. High prevalence of B. hominis and hookworm in this group might reflect their personal hygiene. The majority of the privates originally came from the Northeast, the part of Thailand where people have lower socioeconomic levels. Compared to the group of officers and their families who had lower prevalence of B. hominis infection, most of this latter group had higher education and better hygienic conditions. This agrees with a recent report that showed the association between the infection and lower standards of personal hygiene (Nimri and Batchoun, 1994). Although a few reports found that B. hominis infection was common in the group of over 30 years old (Kain et al, 1987; Doyle et al, 1990), our study showed the highest prevalence in the age group 21 to 30 years old. However, using multivariate analysis, blastocystosis was not significantly associated with age and rank. Generally it has been assumed that B. hominis is transmitted by the same route as other intestinal protozoa (Kain et al, 1987; Zierdt, 1991; Boreham and Stenzel, 1993; Nimri and Batchoun, 1994; Singh et al 1995). Waterborne transmission was speculated since the westerners who were infected by B. hominis usually had a history of drinking untreated water during traveling (Kain et al, 1987). However there is no statistical study to support this hypothesis. Our cross-sectional study showed that an association between blastocystosis and the quality of drinking water was statistically significant. The group of people consuming untreated (neither filtered nor boiled) water, with no respect to the source of water (chlorinated tap or rain water), had significantly greater risk of B. hominis infection. In this area, drinking water was mainly from chlorinated tap and rain water with or without further treatment by filtration or boiling. For the officers and their families, the choice of drinking water and its treatment depended on individual decision. In contrast, privates had only 2 choices of drinking water while they were in this army division. One of the available drinking water sources was chlorinated tap water without any further treatment. It is possible that B. hominis infection might be transmitted via tap water since a speculated transmissionable cyst form can survive up to 19 days in water at normal temperature (Moe et al, 1996). Moreover, a recent study showed resistance of B. hominis cysts to chlorine at the standard concentration (Zaki et al, 1996). Another source of drinking water for the privates was rain water. To collect it, rain water from the roof of each building was transferred through the pipes and kept in the tanks. Thus everything on the roof contaminated the water. To determine whether tap and/or rain water is the source of infection, samples of water in this area have to be further examined. We also collected and examined droppings of pigeons since there were pigeons living on the roofs in considerable numbers. Organisms similar to Blastocystis spp were identified in these samples. Blastocystis spp are common in a wide range of animals (Boreham and Stenzel 1993) and several reports showed a linkage between close contact with pets and farm animals and human blastocystosis Vol 31 No. 1 March 2000 115

SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH (Garavelli and Scaglione 1989; Dolye et al, 1990). It is still unclear that the organisms isolated from animals are genetically linked to B. hominis in humans. Further characterization of the organsims found in the pigeons droppings has to be done. One of many limitations of the cross-sectional study was that it was not the ideal measure for studies of etiology or transmission of diseases. Here, both cases of blastocystosis and exposure to different types of drinking water were observed simultaneously and true risk factors could not be identified. However this study is useful for controlling disease by improvement of sanitary facilities, especially quality of drinking water in this community. Since B. hominis infection was commonly detected in the privates who shared a common living environment, the other routes of transmission such as person-to-person and foodborne transmission should be considered. Without being proven, several reports have suggested these modes of transmission in blastocystosis (Garavelli and Scaglione, 1989; Casemore, 1990; Libanore et al, 1991). The role of B. hominis as a pathogen in humans is still controversial. Many researchers have shown that B. hominis is responsible for nonspecific gastrointestinal symptoms (Sheehan et al, 1986; Kain et al 1987; Hussain Qadri et al, 1989; Doyle et al, 1990; Sinniah and Rajeswari, 1994; Shlim et al, 1995). However most of the patients who had B. hominis infection were asymptomatic. In this study, among the B. hominis infected persons, only 2 of them had diarrhea during the investigation (data not shown). However we cannot conclude that B. hominis was the causative agent since we did not rule out other possible causes. Moreover we found no differences between B. hominis infected group and non-infected group in terms of other presenting symptoms such as abdominal pain, nausea and vomiting (data not shown). In order to explain these findings, factors influencing their pathogenicity have been investigated. Some investigators suggested that B. hominis infection resolved spontaneously (Markell, 1995; Shlim et al, 1995). It has been shown that the selflimiting nature of B. hominis infection is due to protective immunity. Demonstration of strong antibody response against B. hominis infection has been shown (Zierdt et al, 1995). The pathogenicity of B. hominis may be attributed to host immunity since symptomatic blastocystosis has been reported in immunocom-promised hosts (Henry et al, 1986; Laughon et al, 1988; Church et al, 1992; Cegielski et al, 1993). To show whether host immunity influences the severity of blastocystosis, further control studies need to be done. Several reports suggested that the presentation of B. hominis with a large number could cause gastrointestinal illness (Nimri 1993; Nimri and Batchoun, 1994; Shlim et al, 1995). Asymptomatic presentation of the infected persons in this study might be explained by the number of parasites since we found that no one harbored a significant number of B. hominis (less than 5/hpf). However the association between the number of parasites and pathogenicity has been contradicted by several studies (Kain et al, 1987; Doyle et al, 1990; Shlim et al, 1995). Another possibility to explain the different pathogenic potential might be genetic distinction between subgroups. Extensive genetic variation in B. hominis has been shown by using riboprinting. The organisms with indifferent morphology showed at least seven genetic differences (Clark et al, 1997). The author suggested that some strains of B. hominis could be pathogenic to man. This speculation was supported by the study of the parasite isolates from symptomatic patients. It has been shown that a certain variation of small subunit ribosomal RNA of the organism was associated with colonic lesions (Horiki et al, 1998). In contrast, 2 recent studies showed no association between clinical manifestations and any subgroup differentiated either by riboprinting or isoenzyme patterns (Böhm-Gloning et al, 1997; Gericke et al, 1997). Further investigations with a larger scale need to be done. In conclusion, we showed that B. hominis infection in this community was statistically linked to the quality of drinking water. To verify the exact source of infection, samples of water in this area should be examined. Improvement of sanitary facilities especially quality of drinking water in this community in needed. REFERENCES Ashford RW, Atkinson EA. Epidemiology of Blastocystis hominis infection in Papua New Guinea: age-prevalence and associations with other parasites. Ann Trop Med Parasitol 1992; 86: 129-36. Böhm-Gloning B, Knobloch J, Walderich B. Five subgroups of Blastocystis hominis isolates from symptomatic and asymptomatic patients revealed by re- 116 Vol 31 No. 1 March 2000

BLASTOCYSTOSIS IN THAILAND striction site analysis of PCR-amplified 16S-like rdna. Trop Med Int Health 1997; 2: 771-8. Boreham PFL, Stenzel DJ. Blastocystosis in humans and animals: Morphology, biology, and epizootiology. Adv Parasitol 1993; 32: 1-70. Casemore DP. Foodborne protozoal infection. Lancet 1990; 336: 1427-32. Cegielski JP, Msengi EA, Dukes CS, et al. Intestinal parasites and HIV infection in Tanzanian children with chronic diarrhea. AIDS 1993; 7: 213-21. Church DL, Sutherland LR, Gill MJ, et al. Absence of an association between enteric parasites in the manifestations and pathogenesis of HIV enteropathy in gay men. Scand J Infect Dis 1992; 24: 567-75. Clark CG. Extensive genetic diversity in Blastocystis hominis. Mol Biochem Parasitol 1997; 87: 79-83. Doyle PW, Helgeason MM, Mathias RG, Proctor EM. Epidemiology and pathogenicity of Blastocystis hominis. J Clin Microbiol 1990; 28: 116-121. Garavelli PL, Scaglione L. Blastocystosis. An epidemiological study. Microbiologica 1989; 12: 349-50. Gericke A-S, Burchard G-D, Knobloch J, Walderich B. Isoenzyme patterns of Blastocystis hominis patient isolates derived from symptomatic and healthy carriers. Trop Med Int Health 1997; 2: 245-53. Henry MC, De Clereq D, Lokombe B, et al, Parasitological observations of chronic diarrhoea in suspected AIDS adult patients in Kinshasa (Zaire). Trans R Soc Trop Med Hyg 1986; 80: 309-10. Horiki N, Maruyama M, Fujita Y, Tanaka C, Minato S, Cheng X. Genetic diversity in Blastocystis hominis isolates from symptomatic and asymptomatic individuals. Parasitol Int 1998; 47 (suppl): 210. Hussain Qadri SM, Al-Okaili AG, Al Dayel F. Clinical significance of Blastocystis hominis. J Clin Microbiol 1989; 27: 2407-09. Kain KC, Noble MA, Freeman HJ, Barteluk, RL. Epidemiology and clinical features associated with Blastocystosis hominis infection. Diag Microbiol Infect Dis 1987; 8: 235-44. Laughon BE, Druckman DA, Vernon A, et al, Prevalence of enteric pathogens in homosexual men with and without acquired immunodeficiency syndrome. Gastroenterology 1988; 94: 984-93. Libanore M, Rossi MR, Scaglione L, Garavelli PL. Outbreak of blastocystosis in institution for the mentally retarded. Lancet 1991; 337: 609-10. MacPherson DW, MacQueen WM. Morphological diversity of Blastocystis hominis in sodium acetate-acetic acid-formalin-preserved stool samples stained with iron hematoxylin. J Clin Microbiol 1994; 32: 267-8. Markell EK. Is there any reason to continue treating Blastocystis infections? Clin Infect Dis 1995; 21: 104-105. Moe KT, Singh M, Howe J, et al, Observations on the ultrastructure and viability of the cystic stage of Blastocystis hominis from human feces. Parasitol Res 1996; 82: 439-44. Nimri LF. Evidence of an epidemic of Blastocystis hominis infections in preschool children in northern Jordan. J Clin Microbiol 1993; 31: 2706-8. Nimri L, Batchoun R. Intestinal colonization of symptomatic and asymptomatic schoolchildren with Blastocystis hominis. J Clin Microbiol 1994; 32: 2865-6. Sheehan DJ, Raucher BG, McKitrick JC. Association of Blastocystis hominis with signs and symptoms of human disease. J Clin Microbiol 1986; 24: 548-550. Shlim DR, Hoge CW, Rajah R, Rabold JG, Echeverria P. Is Blastocystis hominis a cause of diarrhea in travelers? A prospective controlled study in Nepal. Clin Infect Dis 1995; 21: 97-101. Singh M, Suresh K, Ho LC, Ng GC, Yap EH. Elucidation of the life cycle of the intestinal protozoan Blastocystis hominis. Parasitol Res 1995; 81: 446-50. Sinniah B, Rajeswari B. Blastocystis hominis infection, a cause of human diarrhea. Southeast Asian J Trop Med Public Health 1994; 25: 490-3. Zaki M, Zaman V, Sheikh NA. Resistance of Blastocystis hominis cysts to chlorine. J Pakistan Med Ass 1996; 46: 178-9. Zaman V. Morphology of the cysts of Blastocystis hominis. Southeast Asian J Trop Med Public Health 1994; 25: 790-1. Zaman V. The diagnosis of Blastocystis hominis cysts in human faces. J Infect dis 1996; 33: 15-6. Zierdt CH. Blastocystis hominis-past and future. Clin Microbiol Rev 1991; 4: 61-79. Zierdt CH, Zierdt WS, Nagy B. Enzyme-linked immunosorbent assay for detection of serum antibody to Blastocystis hominis in symptomatic infections. J Parasitol 1995; 81: 127-9. Vol 31 No. 1 March 2000 117